Surgery is the primary treatment of colorectal cancer leading to five-year survival rates of 90 to 40 percent depending on the state of tumour progression from Dukes Stage A to C. Conventional adjuvant therapy that includes radiation therapy and chemotherapy has been able to reduce the death rates further by approximately 30 percent (1). Despite these achievements cancer of the colon and rectum is one of the major causes of death in human cancer. Immunological therapy has been extensively attempted. However, colon cancer has generally been resistant to immunotherapy and is considered to be of low immunogenicity. Patients with colon cancer neither respond to IL-2 treatment or adoptive transfer of in vitro cultured tumour infiltrating lymphocytes otherwise active in patients with immunogenic malignancies such as melanoma. Most encouraging however, Riethmüller et al. reported a 32 percent decreased seven-year death rate for Dukes Stage C colorectal cancer treated after primary tumour resection with a naked murine mAb directed to a tumour and normal epithelial associated antigen (Ep-CAM) (2), indicating that other immunotherapeutic modalities could be effective.
A significant improvement of adjuvant immunotherapy and of the treatment of more advanced stages of cancer should require a more potent effector mechanism than provided by a naked mAb. In principle, an increased potency should require an increased tumour selectivity of the targeting antibody.
The limited number of colon cancer associated antigens defined today have been discovered using hybridoma produced murine mAbs resulting from xenogenic immunisations with human tumours (3).
The use of large phage display libraries for the identification of novel tumour-associated antigens can be expected to significantly speed up the process of finding target molecules useful for tumour immunotherapy and diagnosis. Such identification of target molecules could be accomplished by the selection and screening of antibody phage libraries on cultured tumour cells and tissue sections to generate specific reagents defining in vitro and in vivo expressed antigens (4). The phage display technology has been established as an efficient tool to generate monoclonal antibody reagents to various purified antigens, and the construction and successful selection outcome from immune, naive and synthetic antibody phage libraries have been described in several studies (5).
Non-immune libraries are favourable with respect to their general applicability, making unique libraries for every single target unnecessary. On the other hand, sufficiently large and high quality non-immune libraries are difficult to construct and a target discovery process using these libraries should require efficient subtractive selection methods when based on complex antigens.
A phage library of a more moderate size has now been constructed from a near human primate immunised with complex human antigens. This represents an approach that takes advantage of an in vivo pre-selected repertoire. Such libraries should be enriched for specificities to tumour specific epitopes in a reduced background reactivity to xenogeneic antigens (6). Furthermore, as compared to the mouse, primate antibodies demonstrating close sequence homology with human antibodies should not be immunogenic in man (7).
Novel primate antibodies from a phage library that define selectively expressed colon cancer associated antigens have now been identified. The therapeutic potential, demonstrated by T cell mediated killing of cultured colon cancer cells coated with two of these antibodies fused to engineered superantigens, is comparable with superantigens fused to murine Fab fragment specific for colon cancer associated antigens such as EP-CAM, for which there has previously been established the therapeutic capacity in experimental systems (8).
There is also provided a method for efficient positive and subtractive cell selection of phage antibodies that should facilitate future identification of novel phenotype specific antigens including tumour associated antigens using antibodies from large phage libraries.